Food Chemistry 127 (2011) 1680–1685

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Food Chemistry

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Physicochemical and release characterisation of garlic oil-b-cyclodextrininclusion complexes

Jing Wang ⇑, Yanping Cao ⇑, Baoguo Sun, Chengtao WangCollege of Chemistry and Environment Engineering, Beijing Technology and Business University, 11 Fucheng Road, Beijing 100048, PR ChinaBeijing Higher Institution Engineering Research Center of Food Additives and Ingredients, 11 Fucheng Road, Beijing 100048, PR China

a r t i c l e i n f o a b s t r a c t

Article history:Received 26 August 2010Received in revised form 7 January 2011Accepted 7 February 2011Available online 25 February 2011

Keywords:b-CyclodextrinGarlic oilInclusion complexControlled releaseWater solubility

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.02.036

⇑ Corresponding authors. Address: College of CEngineering, Beijing Technology and Business Univers100048, PR China. Tel.: +86 10 68985378; fax: +86 10

E-mail addresses: jingw810@yahoo.com (J. WanCao).

Garlic oil (GO), rich in organosulphur compounds, has a variety of antimicrobial and antioxidant activi-ties, however, its volatility and low physicochemical stability limit its application as food functionalingredients. The aim of this study was to investigate the physicochemical and release characterisationof inclusion complexes of GO in b-cyclodextrin (b-CD). The formation of GO/b-CD inclusion complexwas demonstrated by different analytical techniques including Fourier transform-infrared spectroscopy,differential scanning calorimetry and X-ray diffractometry. The stoichiometry of the complex was 1:1.The calculated apparent stability constant of GO/b-CD complex was 1141 M�1, and the water solubilityof GO was significantly improved by the phase solubility study. Furthermore, the release of GO fromthe inclusion complex was determined at a temperature range from 25 to 50 �C and in an acidic dissolu-tion medium (pH 1.5), respectively. The release rate of GO from the inclusion complex was controlled.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Garlic (Allium sativum L.) is a widely distributed plant and isused throughout the world not only as a spice and a food, but alsoas a folk-medicine, and many of the beneficial health-relatedbiological effects have been attributed to its characteristic organo-sulphur compounds (Rybak, Calvey, & Harnly, 2004). Steam distil-lation is widely used to extract and condense the volatileorganosulphur compounds in garlic, and the final oily product iscalled garlic oil (GO) (Wu et al., 2002). The compounds of GOmainly are diallyl disulphide (DADS), diallyl trisulphide (DATS), al-lyl propyl disulphide, a small quantity of disulphide and probablydiallyl polysulphide (Pranoto, Salokhe, & Rakshit, 2005). GO isrecognised to be more potent than aqueous extracts of garlic andexhibit a wide range of pharmacological properties including anti-microbial, antidiabetic, antimutagenic, and anticarcinogenic effects(Agarwal, 1996). However, the application of GO in the food indus-try is limited due to its volatility, strong odour, insolubility inwater, and low physicochemical stability (Corzo-Martínez, Corzo,& Villamiel, 2007). Interestingly, theses disadvantages can be ad-dressed by complexation with cyclodextrins (CDs) in aqueous solu-tions. It has been reported that the inclusion complexes of guest

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hemistry and Environmentity, 11 Fucheng Road, Beijing

68985456.g), caoyp@th.btbu.edu.cn (Y.

compounds with CDs can enhance guest stability, improve theaqueous solubility, protect against oxidation, light-induced decom-position, and heat-induced changes, and mask or reduce unwantedphysiological effects, and reduce volatility (Hedges, 1998). Forexample, the natamycin/CDs complexes have allowed a homoge-neous delivery system of natamycin to the shredded cheese surfacewithout the clogging of spray nozzles during cheese production(Koontz & Marcy, 2003).

CDs are non-toxic macrocyclic oligosaccharides, consisting of(a-1,4)-linked a-D-glucopyranose units, with a hydrophilic outersurface and hollow hydrophobic interior (Szente & Szejtli, 2004).CDs are widely used in the food industry as food additives, for sta-bilization of flavours, for elimination of undesired tastes or otherundesired compounds such as cholesterol and to avoid microbio-logical contaminations and browning reactions (Astray, Gonza-lea-Barreiro, Mejuto, Riao-Otero, & Simal-Gándara, 2009). Theyhave the ability to form inclusion complexes with a wide varietyof organic compounds, which enter partly or entirely into the rel-atively hydrophobic cavity of CDs simultaneously expelling thefew high-energy water molecules from inside (Karathanos, Mour-tzinos, Yannakopoulou, & Andrikopoulos, 2007). The most commonCDs used as formulation vehicles are a-, b- and c-CDs containingsix, seven and eight glucopyranose units, respectively. Amongstthe CDs, b-CD is widely used since its cavity size is suitable forcommon guests with molecular weights between 200 and 800 g/mol and also due to its availability and reasonable price (Waleczek,Marques, Hempel, & Schmidt, 2003). Hadaruga, Hadruga, Rivis,Gruia, and Pinzaru (2007) have reported the thermal and oxidative

J. Wang et al. / Food Chemistry 127 (2011) 1680–1685 1681

stability of the A. sativum L. bioactive compounds/a and b-CD nano-particles and discussed that the DADS and DATS were suitablyencapsulated in a higher concentration. It has also been reportedthat the molecular inclusion complexes could be successfullyformed by hydrogen bonding between GO and b-CD and animprovement of stability, solubility, and bioavailability of the guestmolecule in the inclusion complex was obtained (Ayala-Zavalaet al., 2008). Stellenboom, Hunter, Caira, Bourne, and Barbieri(2009) described the preparation of the inclusion complex formedbetween the allicin mimic S-p-tolyl t-butylthiosulphinate and b-CDby both kneading and co-precipitation methods. Bai et al. (2010)have recently reported that two major GO components of DADSand DATS had higher water solubility caused by hydroxypropylb-cyclodextrin (HP-b-CD) and DATS was better suited to be encap-sulated by HP-b-CD compared to DADS.

However, to the best of our knowledge, there are, so far, few re-ports on the inclusion complex of GO and b-CD in the scientific lit-erature. In this study, the inclusion complex of GO/b-CD wasprepared by the co-precipitation method and the formation ofthe inclusion complex of GO with b-CD was analysed by differentanalytical techniques including UV–visible spectroscopy, Fouriertransform-infrared spectroscopy (FT-IR), differential scanning calo-rimetry (DSC) and X-ray diffractometry (XRD). Furthermore, thesolubility and release characterisation of the inclusion complexesof GO in b-CD were determined.

2. Material and methods

2.1. Materials

Garlic oil (GO) (purity > 90%, containing three major constitu-ents, about 35% of diallyl disulphide (DADS), 42% of diallyl trisul-phide (DATS), and 16% of diallyl sulphide), stored at 4 �C, waspurchased from Xunyang Ltd. Co., Guangdong, China. b-cyclodex-trin (b-CD) was purchased from Sinopharm Chemical Reagent Bei-jing Co., Ltd., Beijing, China. All other chemicals and solvents wereof analytical grade.

2.2. Preparation of GO and b-CD Complex

The complex of GO and b-CD was prepared by using a co-pre-cipitation method described by Ayala-Zavala et al. (2008) withminor modification. Briefly, five grams (±0.01) of b-CD was dis-solved in 100 ml of an ethanol to distilled water (1:2, v/v) mixtureand maintained at 60 �C on a hot plate. After cooling the b-CD solu-tion to 40 �C, a portion of 0.5 g of GO dissolved in ethanol (1:1, v/v)was then slowly added to the solution with continuous agitation.The resultant mixture was treated by ultrasonic cleaners at 90 Wfor 4 h. The final solution was maintained overnight at 4 �C. Thecold precipitated GO/b-CD complex was recovered by vacuum-fil-tration. The precipitate was washed twice with 30% ethanol solu-tion to clear GO which was absorbed on the surface of b-CD anddried in a vacuum oven at 40 �C for 4 h until the weight kept con-stant. The final dry complex powders were stored in an airtightglass desiccator at room temperature. The inclusion ratio of GOwas calculated as follows:

Inclusion ratioð%Þ ¼ GO content of inclusion complexðmgÞ½500ðmgÞ= � � 100 ð1Þ

The total recovery was calculated according to the followingequation:

Total recovery ð%Þ ¼ Recovered powder=Initialðb� CD

þ GOÞ � 100 ð2Þ

2.3. Preparation of GO and b-CD physical mixture

b-CD was pulverised in ceramic mortars. The calculatedamounts of both compounds were weighted out at a mass ratioof 10:1 and mixed together by a spatula until a homogeneous mix-ture was obtained.

2.4. Determination of GO in the inclusion complex

GO (5.0 mg) was dissolved into anhydrous ethanol (50 ml). 1.0,2.0, 3.0, 4.0, 5.0 and 6.0 ml solution were taken out and made up to25 ml with ethanol, respectively. These samples were analysed bya UV–visible recording spectrophotometer (Rayleigh AnalyticalInstruments, Beijing, China), monitoring the absorbance at217 nm. The concentration (X) and the absorbance (Y) of GO hada good relationship, when the concentration of GO was <60 lg/ml. The regression equation was as follows:

Y ¼ 0:00859X þ 0:06723; R ¼ 0:9996 ð3Þ

A 20–30 mg of the inclusion complex sample and 30 ml of eth-anol were added to a 50 ml stoppered conical flask bathed in anultrasonic wave cleaner. GO was extracted by ethanol from theinclusion complex for 10 min in the ultrasonic condition. Thesupernatant containing GO was obtained by centrifugation at2500 rpm for 10 min. The content of GO in ethanol was determinedusing ultraviolet spectrophotometry at 217 nm by the calibrationcurve of GO.

2.5. Physicochemical characterisation

2.5.1. UV–visible spectroscopyThe UV–visible absorption spectra were recorded for GO, b-CD,

their physical mixture and the inclusion complex by using a UV–visible recording spectrophotometer (Rayleigh Analytical Instru-ments, Beijing, China). GO and b-CD were dissolved with cyclohex-ane and water, respectively, at the room temperature. The dryphysical mixture and complex (0.10 g) were added to 10 ml ofcyclohexane, respectively, and the mixture was gently shaken for20 min at room temperature and the solvent phases were sepa-rated from the test tube by decantation. The solutions of GO andb-CD, and the cyclohexane extracts were scanned, respectively, inthe range from 200 to 400 nm to obtain the UV–visible absorptionspectra.

2.5.2. Fourier transform-infrared spectroscopy (FT-IR)The FT-IR spectra of GO, b-CD, their physical mixture and the

inclusion complex were collected between 4000 and 500 cm�1

(Mid infrared region) on a Nicolet Nexus Avater 370 FT-IR spectro-photometer (Nicolet, USA) with 256 scans at a resolution of 4 cm�1.GO was recorded on KBr plates. The physical mixture of b-CD andGO and their inclusion complex were ground with spectroscopicgrade potassium bromide (KBr) powder and then pressed into1 mm pellets (2 mg of sample per 200 mg dry KBr). A blank KBrdisc was used as background. FT-IR spectra were smoothed andthe baseline was corrected automatically using the built-in soft-ware of the spectrophotometer (OMNIC 3.2).

2.5.3. Differential scanning calorimetry (DSC)DSC analysis was carried out for GO, b-CD, their physical mix-

ture and the inclusion complex with a Mettler-Toledo DSC821 dif-ferential calorimeter calibrated with indium (Mettler-Toledo S. P.A., Milan, Italy). Each sample (3–5 mg) was heated in a crimpedaluminium pan at a scanning rate of 5 �C/min between 30 and300 �C temperature range under a nitrogen flow of 40 ml/min. Anempty pan sealed in the same way was used as reference. Repro-ducibility was checked by running the sample in triplicate.

1682 J. Wang et al. / Food Chemistry 127 (2011) 1680–1685

2.5.4. X-ray diffractometry (XRD)The X-ray powder diffraction patterns were obtained with a

XRD-6000 X-ray Diffractometer (Shimadzu, Japan) using a Ni-fil-tered, Cu Ka radiation, a voltage of 40 kV and a 30 mA current. b-CD the physical mixture of GO and b-CD and their inclusion com-plex were previously dried for 24 h at 110 �C. Each dried powderwas measured in the 2h angle range between 10 and 80� with ascan rate of 8�/min and a step size of 0.02�. All samples were ana-lysed in triplicate.

2.6. Phase solubility of GO/b-CD complex

Phase solubility studies were carried out according to the meth-od described by Higuchi and Connors (1965). Excess amounts ofGO were added to 10 ml of water solution of b-CD at differentconcentrations ranging from 0 to 9.5 mM. The mixtures were son-icated in an ultrasonic bath for 1 h in the dark at room temperatureand left in the dark for 24 h. After equilibrium was reached, thesample was centrifuged at 3000 rpm for 10 min. The resulting solu-tion was then filtered through a 0.45 lm hydrophilic membranefilter. A small volume of the filtrates was withdrawn and deter-mined for GO at 217 nm by using a UV–visible recording spectro-photometer (Rayleigh Analytical Instruments, Beijing, China). Allsamples were prepared in triplicate. The phase solubility profileswere obtained by plotting the solubility of GO vs. the concentrationof b-CDs. The apparent stability constant, Kc, of GO and b-CD com-plex can be calculated from the slope and the intercept of the linearsegment of the phase solubility line, according to the followingequation:

Kc ¼ k=S0ð1� kÞ ð4Þ

where S0 is the intrinsic solubility of GO in deionized water in theabsence of b-CD and k is the slope of the straight line.

2.7. Release of GO from the complex of GO/b-CD

2.7.1. Release of GO from the inclusion complex at differenttemperature

The release characterisation of GO/b-CD inclusion complex at atemperature range from 25 to 50 �C was determined according tothe method described by Chang, Leung, Lin, and Hsu (2006). Briefly,the release of GO from the complexes at the incubation processwas estimated by measuring the time course of the weightWm(t) of the complexes placed in an Infrared Moisture Determina-tion Balance (IMDB) (AD-4715, A&D Engineering Inc., USA) at 25,37 and 50 �C, respectively. Here, t is the incubation time. Even tinyamounts of vaporisation of solvent could be detected by IMDB, as itis commonly used to determine the water content of fibres. Thesample in the open box of IMDB was heated by using infrared setat desired temperatures. Temperature and weight of the samplewere measured continuously and recorded automatically. The oilrelease content was defined as:

wð%Þ ¼ ½ðWm �WmðtÞÞ=ðWm �W0Þ� � 100 ð5Þ

where W0 denotes the weight of the complexes measured aftercomplete evaporation of GO at 80 �C for 6 h.

2.7.2. In vitro release studyThe in vitro release of GO from the inclusion complex was deter-

mined according to the method described by Haroun and El-Halawany (2010). Briefly, 100 mg GO/b-CD inclusion complexwas placed in 25 ml of an acidic dissolution medium containing0.05 M sodium chloride adjusted to pH 1.5 with HCl at 37 �C. Thestudy was carried out in a shaking water-bath incubator recipro-cating motion (100 rpm). At periodic intervals, samples of the re-lease medium were taken out and spectrometrically assayed for

the amount of GO released at 217 nm, and the volume was re-placed with the fresh acidic dissolution medium (pH 1.5) after eachestimation. These studies were carried out in triplicate. The datarepresent the average from three independent experiments.

2.8. Statistical analysis

The obtained data were expressed as the mean ± standard devi-ation of triplicate determinations. Data were analysed by an anal-ysis of variance (P < 0.05) and the means separated by Duncan’smultiple range test. Statistical analysis was performed using thesoftware STATISTICA 6.0.

3. Results and discussion

3.1. Preparation of GO/b-CD complex

In recent years, b-CD has gained appreciable acceptanceamongst the various types of cyclodextrins. The inclusion com-plexes of b-CD have been successfully used to improve solubility,chemical stability and bioavailability of a number of poorly solublecompounds. Various known methods used for the formation of theinclusion complexes like co-precipitation, neutralisation, kneading,spray drying, freeze-drying, solvent evaporation, and ball-millingand sealed-heating in the laboratory have been widely reported(Yamada et al., 2000). The preparation of inclusion complex iswidely performed using the co-precipitation method in the labora-tory, which has the advantage of easy observation of the complexforming and the guest disappearing during the inclusion (Hedges,1998). In this study, the co-precipitation method was selected toprepare the GO/b-CD inclusion complex. The content of GO in theGO/b-CD complex was 10.5 ± 0.6%, and the inclusion ratio of GOwas 90.3 ± 0.9%, and the total recovery was 78.2 ± 2.3%. Hadarugaet al. (2007) reported that the nano-encapsulation yields of A. sat-ivum L. bioactive compounds/a- and b-CD were >60%, the higherones being obtained for the case of b-CD. Ayala-Zavala et al.(2008) reported that the microcapsule yield at 12:88 (w/w) ratioof GO to b-CD was 94.58%, and the total volatile load was 20.24%.The driving forces between CDs and drugs which have been pro-posed to justify the complex formation are hydrogen bonds, vander Waals forces, hydrophobic interactions and the release of‘‘high-energy water’’ molecules from the cavity (Salústio, Feio, Fig-ueirinhas, Pinto, & Marques, 2009). Del Toro-Sánchez et al. (2010)reported that hydrogen bonds and hydrophobic interactions weredetected between the thyme essential oil constituent and b-CDby IR and 1H NMR spectroscopy.

3.2. Physicochemical characterisation of GO/b-CD complex

3.2.1. UV–visible spectroscopy analysisUV–visible spectroscopy is an important tool to study the com-

plexation of GO with b-CD. In this study, the UV–visible absorptionspectra were recorded for GO, b-CD, their physical mixture and theinclusion complex (Figures not shown). b-CD in water had no UVabsorption. In the spectrum of GO in cyclohexane solution, the kmax

value was found at 217 nm, which was ascribed to the linear diallylgroup in GO. The spectrum of the cyclohexane extract of the phys-ical mixture of GO and b-CD was identical with that of GO. How-ever, the UV absorption peak was not observed in the spectrumof the cyclohexane extract of GO/b-CD complex. Interestingly, theUV absorption peak at 217 nm occurred in the spectrum of cyclo-hexane extract of the hydrolysate of the GO/b-CD complex treatedwith 0.1 M HCl. These results indicated that GO was capable offorming an inclusion complex with b-CD.

J. Wang et al. / Food Chemistry 127 (2011) 1680–1685 1683

3.2.2. FT-IR analysisFT-IR is a useful technique used to confirm the formation of an

inclusion complex. The FT-IR spectra of GO, b-CD, the physical mix-ture of GO and b-CD, GO/b-CD inclusion complex are presented inFig. 1. The FT-IR spectrum of b-CD (Fig. 1A) showed prominentabsorption bands at 3390 cm�1 (for O–H stretching vibrations),2929 cm�1 (for C-H stretching vibrations), 1648 cm�1 (for H–O–Hbending), 1156 cm�1 (for C–O stretching vibration) and 1028cm�1 (C–O–C stretching vibration). The FT-IR spectrum of GO(Fig. 1B) consisted of the prominent absorption bands of asymmet-ric stretching vibration of @CH2 (3080 cm�1), of C–H stretching(3008 cm�1), of symmetrical stretching vibration of @CH2 (2977cm�1), and of –CH2– stretching (2904 cm�1). The very intense peakat 1634 cm�1 is attributed to C@C stretching vibration of the allylgroup. The double peak at 1423–1398 cm�1 may be assigned tothe stretching –CH2– group while CH2@CH– stretching is shiftedto 1216 cm�1. The other very intense peak at 918 cm�1 is attrib-uted to C–S–C stretching vibration. Additionally, the IR spectrumdisplayed the S–C absorption between 800 and 700 cm�1 and S–Sabsorption located between 500 and 400 cm�1. The FT-IR spectrumof the physical mixture (Fig. 1C) showed approximate superimpo-sition of the individual patterns of both b-CD and GO, however, theFT-IR spectrum of the GO/b-CD inclusion complex showed no fea-tures similar to pure GO (Fig. 1D). The bands located at 3080, 3008,2977, 2904, 1634, 1423, 1216, and 918 cm�1 of GO had totally dis-appeared. The GO bands are almost completely obscured by veryintense and broad b-CD bands. However, the absorption bands at3390 and 2929 cm�1 of b-CD were shifted toward these lower fre-quencies at 3382 and 2923 cm�1 of GO/b-CD, respectively. Thesechanges may be related to the formation of intra-molecular hydro-gen bonds between GO and b-CD.

5001000150020002500300035004000

Wavenumbers (cm-1)

A

B

C

D

%T

Fig. 1. FT-IR spectra of b-CD (A), GO (B), GO and b-CD physical mixture (C) and GO/b-CD complex (D).

3.2.3. DSC analysisDSC can be used for the recognition of inclusion complexes.

When guest molecules were embedded into b-CD cavities, theirmelting, boiling or sublimating points generally shifted to differenttemperature or disappeared (Marques, Hadgraft, & Kellaway,1990). The thermal curves of GO, b-CD, the physical mixture ofGO and b-CD, and GO/b-CD are shown in Fig. 2. The thermogramof b-CD showed a wide endothermic peak at about 127 �C(Fig. 2A). The broad endothermic peak was related to dehydrationof water molecules that bind to cyclodextrin molecules (Kohata,Jyodoi, & Ohyoshi, 1993; Marini, Berbenni, Bruni, Giordoano, & Vil-la, 1996). Two exothermic peaks observed at 186 and 223 �C for GOare related to its oxidation (Fig. 2B). Both of peaks at about 104 and118 �C were observed for the case of the physical mixture of GOand b-CD (Fig. 2C). This might be due to the elimination of includedwater molecules with different strengths of interaction with theCD ring (Kohata et al., 1993). DSC curve of the physical mixtureof GO and b-CD was a superimposition of individual componentsof GO and b-CD. A different pattern was observed in the thermo-gram of the GO/b-CD complex (Fig. 3D). The endothermic peak atabout 127 �C originally in the b-CD is slightly shifted to a highertemperature of 131 �C for the inclusion complex system, whichcan be explained on the basis of a major interaction between GOand b-CD. The two exothermic peaks associated with oxidation ofGO were not present in the DSC scan of the GO/b-CD complex, indi-cating that GO is protected from oxidation, being inside the b-CDcavity, and offering an indirect proof of GO’s inclusion.

3.2.4. XRD analysisXRD is a useful method for the detection of CD complexation in

powder or microcrystalline states. The diffraction pattern of thecomplex is supposed to be clearly distinct from that of the super-imposition of each of the components if a true inclusion complexis formed (Veiga, Teixeira-Dias, Kedzierewicz, Sousa, & Maincent,

30 60 90 120 150 180 210 240 270 300Temperature (°C)

Exo

them

ic (

mW

/mg)

A

B

C

D

Fig. 2. DSC thermograms of b-CD (A), GO (B), GO and b-CD physical mixture (C) andGO/b-CD complex (D).

0

200400

600

8001000

1200

1400

0

200

400

600

800

1000

10 11 12 13 14 15 16 17 18 19 20 21 22 23

2θ (°)

10 11 12 13 14 15 16 17 18 19 20 21 22 23

2θ (°)

10 11 12 13 14 15 16 17 18 19 20 21 22 23

2θ (°)

0

200

400

600

800

I (C

ount

s/Se

c)I

(Cou

nts/

Sec)

I (C

ount

s/Se

c)

Fig. 3. Powder X-ray diffraction patterns of b-CD (A), GO and b-CD physical mixture (B) and GO/b-CD complex (C).

1684 J. Wang et al. / Food Chemistry 127 (2011) 1680–1685

1996). The peaks of a sample were intense and sharp, indicating itscrystalline nature. As shown in Fig. 3, some sharp peaks at the dif-fraction angle of 2h 10.66, 12.46, 19.54, 20.70 and 22.70� are pres-ent in the X-ray diffractogram of b-CD powder (Fig. 3A) and itsuggests that the powder is present as a crystalline material. Theb-CD crystallinity peaks were still detectable in the physical mix-ture with GO (Fig. 3B), however, these crystallinity peaks originallyin the b-CD sample disappeared, and a few new sharp peaks at adiffraction angle of 2h 10.08, 12.06, 14.62, 15.48, 17.74, 18.62and 18.90� appeared in the X-ray diffractogram of the complexsample (Fig. 3C), suggesting the formation of the GO/b-CD.

3.3. Phase solubility of GO/b-CD complex

The stoichiometry of the GO/b-CD complex was determined bythe solubility technique. A linear relationship was obtained be-tween the amount of GO solubilised and the concentration of b-CD in solution, which was classified as a typical AL-type. Theregression equation was as follows:

Y ¼ 0:4891X þ 0:0009; R2 ¼ 0:9982 ð6Þ

where Y is the concentration (mM) of GO, X is the concentration(mM) of b-CD. According to Higuchi and Connors’s theory (Higuchi& Connors, 1965), this may be attributed to the formation of a 1:1inclusion complex between GO and b-CD. Bai et al. (2010) reportedthat the stoichiometry of the complex of GO with HP-b-CD was 1:1.Tian, Jiang, and Li (2008) reported that the stoichiometry of theinclusion complexes of Salvia sclarea L. essential oil with CDs was1:1. The calculated apparent stability constant of the GO/b-CD com-

plex was 1141 M�1, which indicated that the interactions betweenGO and b-CD are very strong. As compared with the solubility of GOin deionized water in the absence of b-CD, there is a 6.5-fold in-crease in the presence of 9.5 mM b-CD. Waleczek et al. (2003) re-ported that apparent stability constants of 273 M�1 for the pure(�)-a-bisabolol and 304 M�1 for a constituent of the camomileessential oil were determined by phase solubility tests.

3.4. Release of GO/b-CD inclusion complex

The release profiles of GO from the GO/b-CD complex at a tem-perature range from 25 to 50 �C are shown in Fig. 4. Temperaturehad a pronounced effect on the release rate of GO from the com-plex. At room temperature, the GO/b-CD complex was very stable,and no GO release from the complex was observed within thetested time. An obvious increase for the release rate of GO was ob-served when temperature increased. The required times for 50% re-lease rate at 37 and 50 �C were about 32 and 16 h, respectively.After an incubation of 60 h, the release rate of GO reached 75.8%and 100% at 37 and 50 �C, respectively. These results suggestedthat the steric hindrance of b-CD torus gave protection againstevaporation after GO was included in the cavities of b-CD, andthe release rate of GO depended on the treatment temperatureand treatment time considerably.

The in vitro release profile of GO from the GO/b-CD complex inan acidic dissolution medium (pH 1.5) at 37 �C is shown in Fig. 5.The profile is characterised by an initial fast release phase followedby a delayed release which reaches the plateau level of 100% ofcumulative release rate. The release of GO in the first 2 h was found

Fig. 4. Effect of temperature on the release of GO from the GO/b-CD complex.

0

20

40

60

80

100

0 2 4 6 8 10 12Time (h)

%C

umul

ativ

e R

elea

se

Fig. 5. In vitro release of GO from the GO/b-CD complex in an acidic dissolutionmedium (pH 1.5).

J. Wang et al. / Food Chemistry 127 (2011) 1680–1685 1685

to be 19.2%. At the end of 4 h, the amount of GO released from thecomplex arrived at about 50.7%. The boost release of GO in aqueoussolution might be mainly attributed to the diffusion and inclusionof GO from the surface and cavities of b-CD. After 12 h, 100% of GOwas released from the inclusion complex in the acidic dissolutionmedium.

4. Conclusions

The results of this study clearly demonstrated that GO could beefficiently complexed with b-CD to form an inclusion complex bythe co-precipitation method in a molar ratio of 1:1. The results ofUV–visible spectroscopy, FT-IR, DSC and XRD demonstrated thatGO/b-CD complex has different physicochemical characteristicsfrom free GO. The aqueous solubility and stability of GO were sig-nificantly increased by inclusion in b-CD. The GO release rate fromthe GO/b-CD complex was controlled.

Acknowledgement

This work was supported by a grant from the National HighTechnology Research and Development Program of China (863 Pro-gram) (No. 2007AA10Z306) and Funding Project for Academic Hu-man Resources Development in Institutions of Higher Learning,Beijing New Century BaiQianWan Talent Project and Beijing NovaProgram (No. 2008B07).

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